Kidney production method

ABSTRACT

The present invention provides a kidney production method including a step of tissue-specifically removing a metanephric mesenchyme of a metanephros of a non-human animal; a step of transplanting, into the metanephros, a kidney precursor cell derived from a non-human animal which is allogeneic or xenogeneic to the non-human animal; and a step of advancing development of the metanephros, which is a step in which the transplanted kidney precursor cell is differentiated and matured to form a part of the kidney.

TECHNICAL FIELD

The present invention relates to a kidney production method. Morespecifically, the present invention relates to a kidney productionmethod, a kidney, a non-human animal, an organ for transplantation, akit for kidney production, a medical drug for kidney regeneration, akidney transplantation method, and a method for treating a kidneydisease. Priority is claimed on Japanese Patent Application No.2016-129393, filed on Jun. 29, 2016, the content of which isincorporated herein by reference.

BACKGROUND ART

Due to aging and expansion of an adaptation disease, dialysis patientsare increasing rapidly. The lives of these patients can be saved bydialysis medical treatment, but because dialysis does not compensate forall kidney functions, cardiovascular diseases of the patients tend toincrease, thereby causing rise in mortality rates. In addition, thedialysis patients are overburdened with time and mental loads, whichmakes it difficult to return to society in many cases.

Furthermore, there are about 2 million patients with the end-stage renaldisease requiring a kidney transplantation, and the number of patientstends to further increase due to the organ donor shortage. Therefore,the end-stage renal disease is a serious medical problem.

At present, various tissue-specific precursor cells or tissue stem cellscan be induced from pluripotent stem cells such as iPS cells and EScells (refer to, for example, Non-Patent Document 1). However, forexample, the kidney is an organ with low self-repairing ability, andtherefore the kidney function depends on a complicated structurecomposed of various kinds of cells. For this reason, current techniqueshave not yet reached a stage where kidneys having complicatedthree-dimensional structures can be regenerated from kidney precursorcells.

CITATION LIST Non-Patent Literature

-   [Non-Patent Document 1] Takasato M, et al., Directing human    embryonic stem cell differentiation towards a renal lineage    generates a self-organizing kidney, Nat. Cell Biol., 16 (1),    118-126, 2014.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a technique forproducing a kidney from kidney precursor cells.

Solution to Problem

The present invention includes the following aspects.

(1) A kidney production method including a step of tissue-specificallyremoving a metanephric mesenchyme of a metanephros of a non-humananimal; a step of transplanting, into the metanephros, a kidneyprecursor cell derived from a non-human animal which is allogeneic orxenogeneic to the non-human animal; and a step of advancing developmentof the metanephros, in which the transplanted kidney precursor cell isdifferentiated and matured to form a part of the kidney.

(2) The kidney production method according to (1), further including astep of tissue-specifically removing an ureteric bud of the metanephros;and a step of transplanting, into the metanephros, a kidney precursorcell derived from a non-human animal which is allogeneic or xenogeneicto the non-human animal.

(3) The kidney production method according to (1) or (2), in which thekidney precursor cell is a cat cell.

(4) The kidney production method according to any one of (1) to (3), inwhich the non-human animal is a pig.

(5) The kidney production method according to any one of (1) to (3), inwhich the non-human animal is a mouse.

(6) The kidney production method according to any one of (1) to (3), inwhich the non-human animal is a cat.

(7) A kidney produced by the production method according to any one of(1) to (6).

(8) A kidney including: a cell derived from a non-human animal; and acell derived from a non-human animal which is allogeneic or xenogeneicto the non-human animal, in which a proportion of the cell derived fromthe non-human animal which is allogeneic or xenogeneic to the non-humananimal is 70% by mass or more.

(9) The kidney according to (8), in which the cell derived from thenon-human animal which is allogeneic or xenogeneic to the non-humananimal is a cat cell.

(10) A non-human animal comprising the kidney according to any one of(7) to (9).

(11) An organ for transplantation including: the kidney according to anyone of (7) to (9); a ureter; and a bladder.

(12) The organ for transplantation according to (11), in which theureter and the bladder are derived from a non-human animal.

(13) A kit for kidney production including: a metanephros of a non-humananimal; a medicine which tissue-specifically removes a metanephricmesenchyme of the metanephros; and a kidney precursor cell derived froma non-human animal which is allogeneic or xenogeneic to the non-humananimal.

(14) The kit for kidney production according to (13), further includinga medicine which tissue-specifically removes an ureteric bud of themetanephros.

(15) A medical drug for kidney regeneration including: a metanephros ofa non-human animal; a medicine which tissue-specifically removes ametanephric mesenchyme of the metanephros; and a kidney precursor cellderived from a non-human animal.

(16) The medical drug for kidney regeneration according to (15), furtherincluding a medicine which tissue-specifically removes an ureteric budof the metanephros.

(17) A kidney transplantation method including: a step of transplantingthe organ for transplantation according to claim 11 or 12 into a body ofan affected animal; and a step of connecting a bladder of the organ fortransplantation to a ureter of the affected animal at a predeterminedtime after the transplantation.

(18) A method for treating a kidney disease, including: a step oftransplanting a metanephros and a cloaca of a non-human animal into abody of an affected animal;

a step of tissue-specifically removing a metanephric mesenchyme of ametanephros; a step of transplanting a kidney precursor cell into themetanephros, in which the transplanted kidney precursor cell isdifferentiated and matured to form a part of a kidney, and the cloaca isdifferentiated and matured to form a bladder so as to connect thebladder to the kidney via a ureter; and a step (b) of connecting thebladder to a ureter of the affected animal at a predetermined time afterthe transplantation of the kidney precursor cell.

Advantageous Effects of Invention

According to the present invention, it is possible to provide atechnique for producing a kidney from kidney precursor cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing a fetus of a mouse at around day11 of embryonic development.

(a) to (c) of FIG. 2 are schematic diagrams for explaining how a kidneyis developed.

FIG. 3 is a photograph showing a result of immunostaining of a kidneytissue sample in Experimental Example 3.

FIG. 4 is a photograph showing a result of immunostaining of a kidneytissue sample in Experimental Example 4.

(a) and (b) of FIG. 5 are photographs of a kidney regenerated in a mousebody in Experimental Example 5.

(a) and (b) of FIG. 6 are microphotographs of tissue section samples ofthe kidney regenerated in the mouse body in Experimental Example 5.

(a) to (c) of FIG. 7 are photographs showing results of immunostainingof a tissue sample of a metanephros in Experimental Example 6.

DESCRIPTION OF EMBODIMENTS

[Kidney Production Method]

FIG. 1 is a schematic diagram showing a fetus of a mouse at around day11 of embryonic development. A kidney is formed through three stages ofpronephros, mesonephros, and metanephros. Among these, the pronephrosand mesonephros are degenerated later. A kidney functioning in an adultbody of mammals is the metanephros.

As shown in FIG. 1 , the kidney is formed by an interaction between anureteric bud and a metanephric mesenchyme surrounding the ureteric bud.The ureteric bud constitutes from collecting ducts to a ureter, andnephron precursor cells contained in the metanephric mesenchyme are theorigin of glomeruli and kidney tubules. In the present specification, akidney development region including the ureteric bud and the metanephricmesenchyme is called a kidney development niche.

In one embodiment, the present invention provides a kidney productionmethod including a step (i) of tissue-specifically removing ametanephric mesenchyme of a metanephros of a non-human animal; a step(ii) of transplanting, into the metanephros, a kidney precursor cellderived from a non-human animal which is allogeneic or xenogeneic to thenon-human animal; and a step (iii) of advancing development of themetanephros, which is a step in which the transplanted kidney precursorcell is differentiated and matured to form a part of the kidney.

In the related art, efficiency of a cell transplant into a kidneydevelopment niche was extremely poor in terms of fixation. On the otherhand, as described later in Examples, according to a production methodof the present embodiment, transplanted kidney precursor cells can beengrafted with high efficiency. In addition, the transplanted kidneyprecursor cells take over a development program of a host andautonomously advance complicated differentiation induction to form apart of the kidney.

A kidney production method of the present embodiment will be describedin more detail with reference to FIG. 2 . (a) to (c) of FIG. 2 areschematic diagrams for explaining how a kidney is developed. Amongthese, (a) of FIG. 2 corresponds to the kidney production method of thepresent embodiment.

In (a) of FIG. 2 , a metanephric mesenchyme of an early kidney of anon-human animal embryo is removed by adding a removing medicine. Theaddition results in a state in which kidney stem cells derived from thehost do not exist, that is, a state in which the niche is empty.Subsequently, new kidney precursor cells (kidney stem cells) aretransplanted into this kidney development niche. Then, the transplantedkidney precursor cells are fixed and take over the development programof the host to proceed the development of the kidney. As a result, thekidney is regenerated by the transplanted kidney precursor cells.

Meanwhile, in (b) of FIG. 2 , a metanephric mesenchyme of an earlykidney of a non-human animal embryo is removed by adding a removingmedicine. The addition results in a state in which kidney stem cellsderived from the host do not exist, that is, a state in which the nicheis empty. In (b) of FIG. 2 , a transplantation of new kidney precursorcells is not carried out. Even if the development of the kidneyprogresses in this state, the kidney will be degenerated.

In addition, in (c) of FIG. 2 , new kidney precursor cells aretransplanted into the metanephric mesenchyme of the early kidney of thenon-human animal embryo while remaining the kidney stem cells derivedfrom the host. However, competition by the existing cells derived fromthe host which occupies the kidney development niche is strong, and thusthe transplanted kidney precursor cells are not fixed. As a result, akidney composed of the cells derived from the host is formed.

Hereinafter, each step of the kidney production method of the presentembodiment will be described in detail.

(Step (i))

In the present step, the metanephric mesenchyme of the non-human animalembryo is tissue-specifically removed. The non-human animal is notparticularly limited, and may be, for example, a pig. Pigs are suitablefor a transplantation into a human because of a size of organs thereof,and genetic modification techniques have also been established.Alternatively, the non-human animal may be a mouse. Mice are easy to usebecause genetic modification techniques and various experimental systemshave been established. Alternatively, the non-human animal may be a cat.Cats are one of the most common animals as a pet, but are an animalhaving a very high incidence of chronic kidney disease. For example,there are reports that about 30% of the cause of cat death is chronickidney failure and 50% or more of cats die or are euthanized due to adeterioration in kidney functions. For this reason, there is a potentialdemand for the kidney transplantation as one of life-prolongingtechniques for cats suffering from kidney disease.

Tissue-specific removal of the metanephric mesenchyme is notparticularly limited as long as the metanephric mesenchyme can betissue-specifically removed, and can be performed by using, for example,a genetic recombination technique.

A more specific method of tissue-specifically removing the metanephricmesenchyme will be described by using a mouse system as an example.Examples of the method of tissue-specific removal of the metanephricmesenchyme include a method in which an iDTR mouse expressing adiphtheria toxin receptor (DTR) in a Cre recombinase activity-dependentmanner is crossed with Six2-Cre mouse in which a Cre recombinase gene isintroduced into downstream of a promoter of Six2; and the diphtheriatoxin is brought into contact with the metanephros tissue of a progenyto be obtained.

The iDTR mouse has a transcription termination sequence flanked by twoloxP sequences upstream of a gene encoding the diphtheria toxinreceptor. For this reason, the diphtheria toxin receptor is notexpressed in this state. However, if the transcription terminationsequence flanked by the two loxP sequences is removed by the Crerecombinase, the diphtheria toxin receptor is expressed.

Since mice do not have the diphtheria toxin receptor, cells of mice arenot killed even if the diphtheria toxin is brought into contact with thecells thereof. However, it is known that when the diphtheria toxin isbrought into contact with cells of a mouse expressing the diphtheriatoxin receptor, the cells are killed. In the above-described example,this phenomenon is utilized.

First, if the iDTR mouse is crossed with the Six2-Cre mouse specificallyexpressing the Cre recombinase in the metanephric mesenchyme, a mousetissue-specifically expressing the diphtheria toxin receptor in themetanephros tissue among progenies to be obtained. Six2 is atranscription factor that is specifically expressed in the metanephricmesenchyme.

Subsequently, when the diphtheria toxin is brought into contact with themetanephros of the above-mentioned mouse, cells specifically in themetanephric mesenchyme are killed, and thus the metanephric mesenchymecan be tissue-specifically removed. That is, the kidney developmentniche can be emptied.

The contact with the diphtheria toxin may be carried out by injectingthe diphtheria toxin into a parent mouse or a fetus of a mouse.Alternatively, the contact may be carried out by extracting themetanephros from a fetus of a mouse, organ-culturing the extractedmetanephros, adding the diphtheria toxin to a medium, and the like.Alternatively, the contact may be carried out by transplanting theextracted metanephros into a para-aortic region or greater omentum of anaffected animal, locally administering the diphtheria toxin into a bodyof the affected animal, and the like. In the present specification,examples of the affected animal include cats, dogs, horses (especiallyrace horses), monkeys, cows, sheep, pigs, goats, rabbits, hamsters,guinea pigs, rats, mice, and the like.

The above-described method is one of methods of tissue-specificallyremoving the metanephric mesenchyme, and can be applied to not only micebut also any non-human animal.

(Step (ii))

Subsequently, in the present step, kidney precursor cells derived from anon-human animal which is allogeneic or xenogeneic to the non-humananimal are transplanted into the metanephros. The step (i) and step (ii)may be carried out in parallel. That is, a time of emptying the nicheand a time of transplanting the kidney precursor cells may overlap. Forexample, the addition of the removing medicine and the transplantationof the kidney precursor cells may be carried out at the same time.

Examples of the kidney precursor cells include kidney precursor cells(kidney stem cells) differentiation-induced from pluripotent stem cellssuch as mesenchymal stem cells (MSCs), iPS cells, and ES cells derivedfrom mammals. The kidney precursor cells may be cells derived from anaffected animal or allogeneic cells whose rejection reaction issuppressed in an affected animal.

It is preferable that the kidney precursor cells be cells derived froman affected animal that is a subject of the transplantation of thekidney after production. Examples of the kidney precursor cellsdifferentiation-induced from mesenchymal stem cells sorted from bonemarrow, adipose tissue, bloodshed, or umbilical cord blood, which isderived from an affected animal. The kidney precursor cells may bekidney precursor cells differentiation-induced from mesenchymal stemcells sorted from bone marrow, bloodshed, or umbilical cord blood of anaffected animal itself. A sorting method may be a method according togeneral surgical medical techniques. It is preferable that the sortedcells be cultivated for 2 to 5 times by selecting optimum conditions. Inaddition, the cultivation may be carried out by using a medium kitexclusive for human mesenchymal stem cells manufactured by CambrexBioScience, or the like for the purpose of continuing the cultivationwhile suppressing the transformation of the mesenchymal stem cells.

If desired, a desired gene may be introduced into the kidney precursorcells using adenovirus, retrovirus, or the like. For example, the genemay be introduced so as to express glial cell line-derived neurotrophicfactor-GDNF for the purpose of assisting kidney formation. This isbecause the mesenchymal tissue immediately before the kidney is formedbecomes to express GDNF, and the ureteric bud expressing c-ret which isa receptor thereof is drawn, thereby completing a first important stepof the kidney development.

As a method of the transplantation, for example, the kidney precursorcells derived from the non-human animal may be injected into themetanephros using an injection needle or the like. The number of kidneyprecursor cells to be transplanted is preferably about 1×10³ to 1×10⁶,for example. Because the transplantation of the kidney precursor cellscan be performed in vitro, skill for the operation is not required, andthe operation is easily performed.

The kidney precursor cells may be cat cells. In this case, the kidney tobe produced is composed of cat cells transplanted into the kidneydevelopment niche. This kidney can be transplanted into a cat that is anaffected animal so as to function. The kidney precursor cells are morepreferably cells in which the rejection reaction with cells of theaffected animal or the affected animal is suppressed.

The kidney precursor cells may be cells derived from the non-humananimal other than cats. Examples of non-human animal other than catsinclude the above-mentioned affected animal. In this case, the kidney tobe produced is composed of cells derived from the non-human animal whichare transplanted into the kidney development niche. This kidney can betransplanted into the non-human animal that is the affected animal so asto function. The kidney precursor cells are more preferably cells inwhich the rejection reaction with cells of the affected animal or theaffected animal is suppressed.

(Step (iii))

Subsequently, in the present step, the development of the metanephrosafter transplanting the kidney precursor cells progresses. As a result,the transplanted kidney precursor cells are differentiated and matured,thereby forming a part of the kidney. In a case where the kidneyprecursor cells are transplanted without extracting the metanephros fromthe embryo, the progression of the metanephros can be carried out byreturning the embryo back to the uterus of the parent animal, performingwhole embryo culture, and the like. Alternatively, in a case where themetanephros is extracted to transplant the kidney precursor cells, theprogression can be carried out by continuing an organ culture of themetanephros. Alternatively, in a case where the metanephros istransplanted into the para-aortic region or greater omentum of theaffected animal to transplant the kidney precursor cells into the bodyof the affected animal, the progression can be carried out by growingthe metanephros as it is.

As a result, as will be described later in Examples, the transplantedkidney precursor cells take over the developmental program of the host,autonomously advance complicated differentiation induction whileinteracting with the remaining ureteric bud, and thereby forming theglomeruli and kidney tubules. In addition, in order to completely removethe kidney precursor cells constituting the metanephric mesenchyme onthe host side, the glomeruli and kidney tubules to be regenerated arecomposed only of cells derived from substantially the transplantedkidney precursor cells.

The term “substantially” means that in a case where the kidney isproduced using the kidney development niche of the non-human animal,mixing of cells other than the transplanted kidney precursor cells isnot excluded. In other words, it is preferable that 70% by mass or moreof the glomeruli and kidney tubules to be regenerated be composed of thetransplanted kidney precursor cells, it is more preferable that 80% bymass or more of the glomerulus and kidney tubules to be regenerated becomposed of the transplanted kidney precursor cells, it is even morepreferable that 90% by mass or more of the glomerulus and kidney tubulesto be regenerated be composed of the transplanted kidney precursorcells, it is still more preferable that 95% by mass or more of theglomerulus and kidney tubules to be regenerated be composed of thetransplanted kidney precursor cells, and it is particularly preferablethat 99% by mass or more of the glomerulus and kidney tubules to beregenerated be composed of the transplanted kidney precursor cells.

The kidney production method of the present embodiment may furtherinclude a step (iv) of tissue-specifically removing an ureteric bud ofthe metanephros; and a step (v) of transplanting, into the metanephros,a kidney precursor cell derived from a non-human animal which isallogeneic or xenogeneic to the non-human animal. In addition, the steps(iv) and (v) may be performed before the steps (i) to (iii) describedabove, or may be performed after the steps (i) to (iii) described above.Furthermore, the step (iii) may further be carried out after the steps(iv) and (v).

As a result, not only the metanephric mesenchyme but also the uretericbud can be replaced with cells derived from the transplanted kidneyprecursor cells. As a result, it is possible to produce the kidneysubstantially consisting only of the cells derived from the transplantedkidney precursor cells derived from the non-human animal. Hereinafter,each step will be described.

(Step (iv))

In the present step, the ureteric bud of the metanephros of thenon-human animal is tissue-specifically removed. The tissue-specificremoval of the ureteric bud is not particularly limited as long as theureteric bud can be tissue-specifically removed, and can be performed byusing, for example, a genetic recombination technique.

For example, it is possible to use a mouse obtained by furtherintroducing a gene encoding a Cre-ER protein into downstream of anureteric bud-specific promoter in the mouse obtained by crossing theiDTR mouse and the Six2-Cre mouse described above. Examples of theureteric bud-specific promoter include a promoter of cytokeratin 8, apromoter of HoxB7, and the like.

Examples of such a mouse include an iDTR mouse having a genotype ofSix2-Cre^(tg/wt) cytokeratin 8-Cre-ER^(tg/wt), an iDTR mouse having agenotype of Six2-Cre^(tg/wt)HoxB7-Cre-ER^(tg/wt), and the like. The term“tg” represents that the type is transgenic, and “wt” represents a wildtype.

The Cre-ER protein is a fusion protein of a Cre recombinase and a mutantestrogen receptor. The above-described mouse expresses Cre-ER in apromoter-dependent manner with respect to cytokeratin 8 or HoxB7, whichis a marker for the ureteric bud.

Although the Cre-ER protein is generally present in the cytoplasm, theCre-ER protein is translocated into the nucleus by binding withtamoxifen, which is an estrogen derivative, and recombines against theloxP sequence. By utilizing the above description, It is possible totamoxifen-dependently adjust a working time of a Cre-loxP system.Therefore, even if Cre-ER and Cre are expressed at the same time, it ispossible to control the expression of Cre-ER activity depending onwhether tamoxifen is administered or not. Cre-ERT, Cre-ERT2, or thelike, which is a modified form of Cre-ER, may be used instead of Cre-ER.

The mouse described above expresses the diphtheria toxin receptorspecifically in the metanephric mesenchyme. In this case, it ispreferable to first perform the steps (i), (ii), and (iii) describedabove. Specifically, the metanephric mesenchyme is removed by bringingthe diphtheria toxin into contact with the metanephros of the mouseembryo. Subsequently, the kidney precursor cells derived from thenon-human animal are transplanted into the metanephros by theabove-described method so that the development of the metanephrosprogresses. As a result, the metanephric mesenchyme is regenerated bythe transplanted kidney precursor cells derived from the non-humananimal.

Subsequently, when tamoxifen is brought into contact with thismetanephros, the diphtheria toxin receptor is expressed specifically inthe ureteric bud. In this case, by bringing the diphtheria toxin intocontact with this metanephros, the ureteric bud can betissue-specifically removed. That is, the kidney development niche canbe emptied.

In the above example of the mouse, the gene encoding the Cre protein isintroduced into downstream of the metanephric mesenchyme-specificpromoter, and the gene encoding the Cre-ER protein is introduced intodownstream of the ureteric bud-specific promoter. However, the geneencoding the Cre-ER protein may be introduced into downstream of themetanephric mesenchyme-specific promoter, and the gene encoding the Creprotein may be introduced into downstream of the ureteric bud-specificpromoter. In addition, Cre-ERT, Cre-ERT2, or the like, which is amodified form of Cre-ER, may be used instead of Cre-ER.

For example, a mouse obtained by further introducing the gene encodingthe Cre protein into downstream of the ureteric bud-specific promoter inthe mouse obtained by crossing the iDTR mouse with the mouse in whichthe gene encoding the Cre-ER protein is introduced into downstream ofthe metanephric mesenchyme-specific promoter, may be used.

More specific examples of the mouse include an iDTR mouse having agenotype of Six2-Cre-ER^(tg/wt)HoxB7-Cre^(tg/wt), and the like. Theterms “tg” and “wt” have the same meanings as those described above.

This mouse expresses Cre-ER specifically in the metanephric mesenchyme.In addition, the diphtheria toxin receptor is specifically expressed inthe ureteric bud. Therefore, in the case of using this mouse, the steps(iv) and (v) are carried out before carrying out the steps (i) to (iii).Specifically, by bringing the diphtheria toxin into contact with themetanephros of the mouse embryo, the ureteric bud can be removed.

The above-described method is one of methods of tissue-specificallyremoving the ureteric bud, and can be applied to not only mice but alsoany non-human animal.

(Step (v))

Subsequently, in the present step, the kidney precursor cells derivedfrom the non-human animal which is allogeneic or xenogeneic to thenon-human animal are transplanted into the metanephros so that thedevelopment of the ureteric bud progresses. As a result, the uretericbud is regenerated by the transplanted human kidney precursor cells. Thestep (iv) and step (v) may be carried out in parallel. That is, a timeof emptying the niche and a time of transplanting the kidney precursorcells may overlap. For example, the addition of the removing medicineand the transplantation of the kidney precursor cells may be carried outat the same time.

Subsequently, the above-described step (iii) is carried out so that thedevelopment of embryos (metanephros) transplanted with the kidneyprecursor cells derived from the non-human animal progresses. As aresult, the transplanted kidney precursor cells derived from thenon-human animal are differentiated and matured, thereby forming a partof the kidney.

As the kidney precursor cells derived from the non-human animal, cellsthat are the same as those described above can be used. In a case wherethe kidney precursor cells have the same origin as the kidney precursorcells in the regeneration of the metanephric mesenchyme described above,not only the glomeruli and kidney tubules but also the collecting ductsand ureter are composed of cells of the same origin in the kidney to beproduced.

Therefore, if the kidney precursor cells are cells derived from theaffected animal or cells in which the rejection reaction with theaffected animal is suppressed, it is possible to produce the kidney withless rejection reaction in a case of transplanting these kidneyprecursor cells into the affected animal.

For example, in a case where the steps (iv) and (v) are performed firstusing the iDTR mouse having the genotype ofSix2-Cre-ER^(tg/wt)HoxB7-Cre^(tg/wt), the steps (i), (ii), and (iii)described above may be subsequently carried out. That is, when tamoxifenis brought into contact with the metanephros of the mouse whose uretericbud has been regenerated by transplanted kidney precursor cells derivedfrom the non-human animal, the diphtheria toxin receptor is expressedspecifically in the metanephric mesenchyme in this case. Therefore, inthis case, by bringing the diphtheria toxin into contact with thismetanephros, the metanephric mesenchyme can be tissue-specificallyremoved in this case. That is, the kidney development niche can beemptied.

The above-described method is one of methods of tissue-specificallyremoving the ureteric bud, and can be applied to not only mice but alsoany non-human animal.

In the kidney production method of the present embodiment, any of theremoval of the metanephric mesenchyme and the removal of the uretericbud may be performed first. In a case of removing the ureteric budfirst, the above-described Cre-loxP system may be appropriatelymodified.

In addition, in the kidney production method of the present embodiment,a system for killing cells can be used without particular limitation aslong as the system can be applied to a target non-human animal speciesand can be operated in a tissue-specific manner or in a time-specificmanner.

Therefore, a configuration in which the cells are killed by a systemother than the diphtheria toxin receptor may be adopted. Examplesthereof include a system that expresses a diphtheria toxin A subunit(DT-A) at downstream of a tissue-specific promoter in the Crerecombinase activity-dependent manner. This system can be applied tonon-human animals which are originally sensitive to the diphtheriatoxin. Examples of such a non-human animal include a pig.

Alternatively, examples thereof include a system that expresses a herpessimplex virus-derived thymidine kinase gene (HSV-TK) which inducesapoptosis by administration of ganciclovir, at downstream of thetissue-specific promoter. In cells expressing HSV-TK, cell death isinduced under administration of ganciclovir.

Alternatively, a system that induces apoptosis by dimerizing Caspase-3,Caspase-8, Caspase-9, and the like by administration of AP20187 may beused.

The kidney production method of the present embodiment is not limited tothe method described above as long as the metanephric mesenchyme or theureteric bud of the metanephros can be tissue-specifically removed, andit is possible to use various genetic recombination systems and acombination of genetic recombination systems.

[Kidney]

In one embodiment, the present invention provides a kidney produced bythe production method described above. The kidney of the presentembodiment is produced from the kidney precursor cells derived from adesired non-human animal by utilizing the kidney development niche andthe developmental program of a host non-human animal. Therefore, thekidney can be transplanted into the affected animal so as to function.

As described above, the kidney of the present embodiment is formed byreplacing the metanephric mesenchyme or the ureteric bud, or boththereof with the transplanted kidney precursor cells derived from thenon-human animal. For this reason, in particular, in a case of thekidney in which both metanephric mesenchyme and ureteric bud arereplaced with the transplanted kidney precursor cells derived from thenon-human animal, this kidney is substantially composed only of cellsderived from the transplanted kidney precursor cells derived from thenon-human animal. For this reason, merely by specifying the cellsconstituting the kidney, it is difficult to specify whether a kidney isthe kidney produced by the above-described production method, or is akidney of an affected animal from which the transplanted kidneyprecursor cells derived from the non-human animal had been prepared.

The kidney includes cells derived from a non-human animal; and cellsderived from a non-human animal which is allogeneic or xenogeneic to thenon-human animal, in which a proportion of the cells derived from thenon-human animal which is allogeneic or xenogeneic to the non-humananimal is 70% by mass or more, more preferably 80% by mass or more, evenmore preferably 90% by mass or more, still more preferably 95% by massor more, and particularly preferably 99% by mass or more.

Examples of the non-human animals include pigs, mice, cats, and the likeas described above. In addition, examples of the cells derived from anon-human animal which is allogeneic or xenogeneic to these non-humananimals include the cells derived from the affected animal describedabove, and the like. Such a kidney can be produced by theabove-described kidney production method.

The kidney of the present embodiment may be formed by replacing thekidney precursor cells derived from the non-human animal, which aretransplanted with the metanephric mesenchyme by the above-describedproduction method, for example. In this case, the collecting ducts andureter derived from the ureteric bud are derived from a host non-humananimal.

Alternatively, the kidney of the present embodiment may be formed byreplacing the kidney precursor cells derived from the non-human animal,which are transplanted with the ureteric bud by the above-describedproduction method. In this case, the glomeruli and kidney tubulesderived from the metanephric mesenchyme are derived from a hostnon-human animal.

Alternatively, the kidney of the present embodiment may be formed byreplacing the kidney precursor cells derived from the non-human animal,which are transplanted with both metanephric mesenchyme and ureteric budby the above-described production method. In this case, the kidney is akidney derived from the kidney precursor cells derived from thenon-human animal, to which substantially all of the glomeruli, kidneytubules, collecting ducts, and ureter are transplanted. However, cellsderived from a host non-human animal may remain in the formed kidney insome cases.

[Organ for Transplantation]

In one embodiment, the present invention provides an organ fortransplantation including any of the kidney, ureter, and bladderdescribed above.

The inventors of the present invention have previously been able togenerate urine by transplantation of regenerated kidneys. However, theinventors have found that generated urine could not be excreted, whichresulted in hydronephrosis, and therefore kidney functions could not bemaintained for a long time. The inventors of the present invention alsohave found that, by transplanting a structure including not only thekidney but also the kidney, the ureter, and the bladder, not onlygeneration of urine but also excretion of the generated urine ispossible.

Therefore, since the organ for transplantation of the present embodimentincludes the kidney, the ureter, and the bladder, even in a case wherethis organ is transplanted into an affected animal, not only generationof urine but also excretion of the generated urine is possible, andkidney functions can be maintained for a long time.

The organ for transplantation of the present embodiment may betransplanted into the para-aortic region or greater omentum of theaffected animal, and may be transplanted into, for example, the spleen(around the splenic artery) of the affected animal. In addition, thebladder constituting the organ for transplantation described above maybe connected to the ureter of the affected animal. In this manner, anorgan structure in which the kidney, a first ureter, a first bladder, asecond ureter, and a second bladder are connected in this order isformed. The second ureter and the second bladder are the ureter andbladder that the affected animal has inherently.

By forming such an organ structure, excretion of the urine generated bythe regenerated kidneys constituting the above-described organ fortransplantation becomes easier.

In the organ for transplantation of the present embodiment, the ureter(first ureter) and the bladder (first bladder) may be derived from thenon-human animal in the kidney production method described above.Because the ureter and bladder generally have low immunogenicity, aproblem is less likely to occur even if the ureter and the bladder arederived from an animal xenogeneic to the non-human animal. The non-humananimal is a host animal which provides the kidney development nicheserving as a scaffold in the kidney production method described above.That is, the organ for transplantation of the present embodiment may becomposed of the kidney formed by the above-described production method,a ureter, and a bladder.

The organ for transplantation of the present embodiment can be extractedfrom a non-human animal, stored, and distributed while being in a statewhere the kidney, ureter, and bladder are connected to each other. Theorgan for transplantation may be frozen for storage and distribution.Alternatively, the organ for transplantation of the present embodimentcan be distributed in a form of a non-human animal having the organ fortransplantation of the present embodiment.

[Kit for Kidney Production]

In one embodiment, the present invention provides a kit for kidneyproduction including a metanephros of a non-human animal; a medicinethat tissue-specifically removes a metanephric mesenchyme of the embryo;and a kidney precursor cell derived from a non-human animal which isallogeneic or xenogeneic to the non-human animal.

By carrying out the above-mentioned kidney production method using thekit of the present embodiment, the kidney can be produced. Examples ofthe non-human animals include pigs, mice, cats, and the like asdescribed above. The metanephros of the non-human animal may be providedin a form of an embryo or may be provided in a form of a parent animalincluding a fetus. In addition, the metanephros may be in a state ofbeing connected to a cloaca which later becomes the bladder. In thiscase, the metanephros forms the kidney and ureter, the cloaca forms thebladder, and therefore a state in which these kidneys, ureters, andbladders are connected is formed. As a result, as described above, theproduced kidneys are capable of not only generating urine but alsoexcreting the produced urine.

In the kit of the present embodiment, examples of the medicine thattissue-specifically removes the metanephric mesenchyme include theabove-mentioned diphtheria toxin, and the like. In this case, examplesof a non-human animal include a non-human animal that expresses thediphtheria toxin receptor specifically in the metanephric mesenchyme bythe Cre-loxP system or the like.

Alternatively, various conditional knockout systems other than thosedescribed above can also be used as long as the metanephric mesenchymecan be tissue-specifically removed thereby. As the medicine fortissue-specifically removing the metanephric mesenchyme, diphtheriatoxin (iDTR system), tamoxifen (Cre-ERT2 system), tacrolimus (Mos-iCsp3system), doxycycline, tetracycline (Tet-On/off system), ganciclovir(herpes virus-derived thymidine kinase gene system), and the like can beused depending on a system to be used.

These conditional knockout systems can be produced using genome editingtechniques and the like, and can tissue-specifically remove cells in thekidney development niche of the non-human animal. In addition, a time atwhich cells are removed can be controlled by administering the medicine.By using these systems, it is possible to spatiotemporal-specificallycontrol the removal of target cells and construct a living scaffoldsuitable for the kidney regeneration.

A method for administering the medicine that tissue-specifically removesthe metanephric mesenchyme is not particularly limited, and may beappropriately selected according to the medicine to be used. Examplesthereof include addition to a medium such as organ culture in vitro,local administration in vivo, intraperitoneal administration,intravenous injection, oral administration, and the like.

In addition, examples of the kidney precursor cells derived from anon-human animal which is allogeneic or xenogeneic to the non-humananimal include the kidney precursor cells derived from the affectedanimal described above, and the like. Examples of the kidney precursorcells include kidney precursor cells differentiation-induced from iPScells, mesenchymal stem cells (MSCs), and the like which are derivedfrom an affected animal; kidney precursor cells differentiation-inducedfrom allogeneic iPS cells or ES cells, in which the rejection reactionin an affected animal is suppressed; and the like. The allogeneic iPScells described above can be obtained from, for example, a cell bank orthe like.

The kidney production may be performed outside the body of the affectedanimal, and then the kidney may be transplanted into the para-aorticregion or greater omentum of the affected animal. Alternatively, thekidney production may be performed in the body of the affected animal.More specifically, the kidney may be produced by transplanting themetanephros of the non-human animal into the para-aortic region orgreater omentum of the affected animal first, emptying the niche of themetanephros in the body of the affected animal, and transplanting thekidney precursor cells. This method is advantageous in that a vascularsystem drawn into the regenerated kidney becomes a blood vessel of theaffected animal, and that a functional kidney can be constructed.

The kit of the present embodiment may further include a medicine thattissue-specifically removes the ureteric bud of the metanephros.Accordingly, it is possible to produce a kidney derived from the kidneyprecursor cells transplanted with not only tissues derived from themetanephric mesenchyme but also tissues derived from the ureteric bud.

Examples of the medicine that tissue-specifically removes the uretericbud include a combination of tamoxifen and diphtheria toxin as describedabove, and the like. In this case, examples of a non-human animalinclude a non-human animal that expresses the diphtheria toxin receptorspecifically in the metanephric mesenchyme by the Cre-loxP system or thelike, and also expresses the diphtheria toxin receptor specifically inthe ureteric bud by administration of tamoxifen.

Alternatively, any of the various conditional knockout systems describedabove can be used to remove the ureteric bud. In this case, the medicinethat tissue-specifically removes the ureteric bud becomes an appropriatemedicine according to an adopted system.

By combining the above-described various conditional knockout systems,it is possible to remove a plurality of target tissues at any timing.For example, after tissue-specifically removing the ureteric bud byusing tamoxifen, the metanephric mesenchyme can be tissue-specificallyremoved by using tacrolimus in a stepwise manner. As a result, almostall of the kidney tissues can be tissues derived from the transplantedkidney precursor cells.

[Medical Drug for Kidney Regeneration]

In one embodiment, the present invention provides a medical drug forkidney regeneration including a metanephros of a non-human animal; amedicine that tissue-specifically removes a metanephric mesenchyme ofthe metanephros; and a kidney precursor cell derived from a non-humananimal.

According to the medical drug of the present embodiment, it is possibleto perform the regeneration of the kidney within the body of theaffected animal as described above. The medical drug of the presentembodiment can be used as follows. First, the metanephros of a non-humananimal is transplanted into the para-aortic region or greater omentum ofthe affected animal. Examples of the affected animal include thosementioned above. As the metanephros of the non-human animal, the samemetanephros as in the above-described kit for kidney production can beused.

Subsequently, the medicine that tissue-specifically removes themetanephric mesenchyme is administered to the affected animal. As themedicine, the same medicine as in the above-described kit for kidneyproduction can be used. It is preferable that the administration of themedicine be appropriately carried out by local administration,intraperitoneal administration, intravenous injection, oraladministration, and the like depending on the medicine to be used.

Subsequently, the kidney precursor cells derived from the non-humananimal are transplanted. Examples of the kidney precursor cells derivedfrom the non-human animal include kidney precursor cellsdifferentiation-induced from iPS cells, mesenchymal stem cells (MSCs),and the like which are derived from an affected animal; kidney precursorcells differentiation-induced from pluripotent stem cells such asallogeneic iPS cells or ES cells, in which the rejection reaction in anaffected animal is suppressed; and the like. Thereafter, the developmentof the transplanted metanephros progresses, thereby producing(regenerating) the kidney composed of the kidney precursor cellstransplanted into the body of the affected animal.

This medical drug of the present embodiment is advantageous in that avascular system drawn into the regenerated kidney becomes a blood vesselof the affected animal, and that a functional kidney can be constructed.

The medical drug for kidney regeneration may further include a medicinethat tissue-specifically removes that is configured totissue-specifically remove the ureteric bud of the metanephros.Accordingly, it is possible to regenerate a kidney derived from thekidney precursor cells transplanted with not only tissues derived fromthe metanephric mesenchyme but also tissues derived from the uretericbud. As the medicine that tissue-specifically removes the ureteric budof the metanephros, the same medicine as in the above-described kit forkidney production can be used.

[Kidney Transplantation Method]

In one embodiment, the present invention provides a kidneytransplantation method including: a step (a) of transplanting the organfor transplantation which has the kidney, ureter, and bladder describedabove into a body of an affected animal; and a step (b) of connecting abladder of the organ for transplantation to a ureter of the affectedanimal at a predetermined time after the transplantation. Examples ofthe affected animal include the same affected animal as mentioned above.

The method of the present embodiment can also be said to be a method fortreating a kidney disease. As the organ for transplantation, thosedescribed above can be used.

By allowing the organ for transplantation which is transplanted in thestep (a) to stand for a predetermined period, the organ fortransplantation grows, and urine production is started. Thepredetermined period may refer to any period up to the organ fortransplantation sufficiently grows, but before occurrence ofhydronephrosis. The predetermined period may be appropriately adjusteddepending on the symptoms and the like of the affected animal, but maybe, for example, 1 to 10 weeks.

Subsequently, the step (b) is performed at a predetermined period oftime (after 1 to 10 weeks) after transplantation. In the step (b), thebladder of the organ for transplantation and the ureter of the affectedanimal are connected. The ureter of the affected animal is connected tothe bladder of the affected animal.

By connecting the bladder of the organ for transplantation and thebladder of the affected animal with the ureter of the affected animal,the urine produced by the organ for transplantation can be excreted tothe bladder of the affected animal. More specifically, the urineproduced by the kidney of the organ for transplantation is excreted tothe bladder of the organ for transplantation through the ureter of theorgan for transplantation, and then is excreted to the bladder of theaffected animal from the bladder of the organ for transplantationthrough the ureter of the affected animal.

By the method of the present embodiment, the urine produced by thekidney of the organ for transplantation can be excreted to the bladderof the affected animal.

[Method for Treating Kidney Disease]

In one embodiment, the present invention provides a method for treatinga kidney disease, including a step (a1) of transplanting a metanephrosand a cloaca of a non-human animal into a body of an affected animal; astep (a2) of tissue-specifically removing a metanephric mesenchyme of ametanephros; a step (a3) of transplanting a kidney precursor cell to themetanephros, which is a step in which the transplanted kidney precursorcell is differentiated and matured to form a part of kidneys, and thecloaca is differentiated and matured to form a bladder so as to connectthe bladder to the kidney via a ureter; and a step (b) of connecting thebladder to a ureter of the affected animal at a predetermined time afterthe transplantation of the kidney precursor cell. Examples of theaffected animal include those mentioned above.

In the method of the present embodiment, the production (regeneration)of the kidney is performed in the body of the affected animal. The step(a2) and the step (a3) may be carried out in parallel. That is, a timeof emptying the niche and a time of transplanting the kidney precursorcells may overlap. For example, the addition of the removing medicineand the transplantation of the kidney precursor cells may be carried outat the same time. Examples of the kidney precursor cells include thesame kidney precursor cells as those for the above-described medicaldrug for kidney regeneration.

When a predetermined period has elapsed from the transplantation of thekidney precursor cells in the step (a3), the transplanted kidneyprecursor cells form the kidney, and urine production is started. Thepredetermined period may refer to any period up to the kidney andbladder sufficiently grow, but before occurrence of hydronephrosis. Thepredetermined period may be appropriately adjusted depending on thesymptoms and the like of the affected animal, but may be, for example, 2to 4 weeks.

Subsequently, the step (b) is performed at a predetermined period oftime (after 2 to 4 weeks) after transplantation. In the step (b), thebladder formed by differentiation and maturation of the cloaca isconnected to the ureter of the affected animal. The ureter of theaffected animal is connected to the bladder of the affected animal.

The treatment method of the present embodiment may further include astep (a2′) in which the ureteric bud of the metanephros istissue-specifically removed. As a result, not only the metanephricmesenchyme but also the ureteric bud can be replaced with cells derivedfrom the transplanted kidney precursor cells derived from the non-humananimal. As a result, it is possible to produce the kidney substantiallyconsisting only of the cells derived from the transplanted kidneyprecursor cells derived from the non-human animal.

In a case of carrying out the step (a2′), any of the step (a2) and thestep (a2′) may be carried out first, but after carrying out either onestep thereof, it is preferable to provide a period of 4 to 7 days beforecarrying out the other step. Between the steps, transplanted kidneyprecursor cells derived from the non-human animal progress thedevelopment program to be differentiated.

The step (a2) and step (a3), or the step (a2′) and step (a3) may becarried out in parallel. That is, a time of emptying the niche and atime of transplanting the kidney precursor cells derived from thenon-human animal may overlap. For example, the addition of the removingmedicine and the transplantation of the kidney precursor cells derivedfrom the non-human animal may be carried out at the same time.

For example, the step (a2) and step (a3) may be carried out at the sametime first, and after a predetermined period, the step (a2′) and step(a3) may be carried out at the same time. Alternatively, the step (a2′)and step (a3) may be carried out at the same time first, and after apredetermined period, the step (a2) and step (a3) may be carried out atthe same time.

EXAMPLES

Next, the present invention will be described in more detail by showingexperimental examples, but the present invention is not limited to thefollowing experimental examples.

Experimental Example 1

(Tissue-Specific Removal of Metanephric Mesenchyme in Mouse Embryo)

The iDTR mouse expressing the diphtheria toxin receptor (DTR) in the Crerecombinase activity-dependent manner was crossed with the Six2-Cremouse in which the Cre recombinase gene was introduced into downstreamof a promoter of Six2. Six2 is a transcription factor that isspecifically expressed in the metanephric mesenchyme.

Subsequently, a mouse that expressed the diphtheria toxin receptorspecifically in the metanephric mesenchyme was selected from an F₁ mouseon embryonic day 13, and the metanephros was extracted. The extractedmetanephros was organ-cultured according to a standard method, anddiphtheria toxin was added to the medium. As a result, Six2-positivekidney precursor cells of the metanephric mesenchyme, in which thediphtheria toxin receptor was expressed were killed. As a result, themetanephric mesenchyme was tissue-specifically removed.

Experimental Example 2

(Transplantation of Kidney Precursor Cells)

The kidney precursor cells derived from a wild-type mouse were preparedfrom the metanephros by a standard method and dissociated into singlecells using a cell dissociation reagent. Subsequently, 1×10⁵ kidneyprecursor cells were transplanted into metanephros tissue from which themetanephric mesenchyme was tissue-specifically removed, which wasprepared in Experimental Example 1. Thereafter, the organ culture of themetanephros tissue was continued.

Experimental Example 3

(Immunostaining 1 of Kidney Tissue)

The metanephros tissue of Experimental Example 2 was fixed with 4%paraformaldehyde after being cultured for 5 days from thetransplantation of the kidney precursor cells, and therefore a tissuesection sample was prepared. Subsequently, Six2 which is a marker of themetanephric mesenchyme and cytokeratin 8 which is a marker of theureteric bud were immunostained, respectively. FIG. 3 is a fluorescencemicroscopy photograph showing the result of immunostaining. Themagnification is 100 times. In FIG. 3 , Six 2-positive cells are derivedfrom the transplanted kidney precursor cells. In addition, cytokeratin8-positive cells are derived from the host animal (F₁ mouse ofExperimental Example 1).

As a result, it was confirmed that, using the ureteric bud derived fromthe F₁ mouse of Experimental Example 1 as a scaffold, the kidneyprecursor cells transplanted in Experimental Example 2 took over thedevelopment program of the host, autonomously advanced complicateddifferentiation induction, and thus formed the metanephric mesenchyme.This result shows that the cells constituting the metanephric mesenchymecan be replaced with the cells derived from the transplanted kidneyprecursor cells by the above-described method.

Experimental Example 4

(Immunostaining 2 of Kidney Tissue)

The kidney precursor cells derived from a transgenic mouse (GFP tgmouse) into which a green fluorescent protein (GFP) gene was introducedwere prepared from the metanephros by a standard method and dissociatedinto single cells using a cell dissociation reagent. Subsequently, 1×10⁵kidney precursor cells were transplanted into metanephros tissue fromwhich the metanephric mesenchyme was tissue-specifically removed, whichwas prepared in the same manner as in Experimental Example 1.Thereafter, the organ culture of the metanephros tissue was continued.

Subsequently, the metanephros tissue was fixed with 4% paraformaldehydeafter being cultured for 7 days from the transplantation of the kidneyprecursor cells, and therefore a tissue section sample was prepared.Subsequently, cytokeratin 8, which is a marker for the ureteric bud, andWilms tumor suppressor protein-1 (WT1), which is a marker of glomerulus,were immunostained, respectively. Subsequently, fluorescence of GFPexpressing the transplanted cells, and immunostained cytokeratin 8 andWT1 was observed with a fluorescence microscope. FIG. 4 is afluorescence microscopy photograph showing the result of immunostaining.The magnification is 200 times. In FIG. 4 , GFP-positive cells andWT1-positive cells are derived from the transplanted kidney precursorcells. In addition, cytokeratin 8-positive cells are derived from thehost animal (F₁ mouse of Experimental Example 1).

As a result, it was confirmed that the transplanted kidney precursorcells derived from the GFP tg mice formed the glomeruli and kidneytubules. This result shows that it is possible to form functional kidneytissue from the kidney precursor cells by the method described above.

Experimental Example 5

(Regeneration of Kidney in Living Body of Mouse)

In the para-aortic region of the wild-type mouse, the cloaca and themetanephros tissue-specifically expressing the diphtheria toxin receptorin the metanephros tissue, which are obtained by crossing the iDTR mousewith Six2-Cre mouse, were transplanted.

Subsequently, by administering the diphtheria toxin, the metanephricmesenchyme of the metanephros was removed, and 1×10⁵ kidney precursorcells derived from the GFP tg mouse were transplanted. Subsequently, amouse transplanted with the metanephros was raised for 7 days.

Subsequently, an abdominal cavity of the above-mentioned mouse wasopened to observe the kidney (regenerated kidney) formed from thetransplanted metanephros.

(a) of FIG. 5 is a photograph of a region where regenerated kidneysexist. The magnification is 15 times. The upper right shows the kidneyof the wild-type mouse (host). The regenerated kidneys exist in a regionsurrounded by a dotted line near the center.

(b) of FIG. 5 is a photograph showing the observation result of thefluorescence of GFP in the same field of view as (a) of FIG. 5 . Themagnification is 15 times. As a result, fluorescence of GFP derived fromtransplanted kidney precursor cells was observed. This result indicatesthat the kidney regeneration was possible in the body of the mouse.

(a) of FIG. 6 is an optical micrograph of a tissue section sampleprepared by fixing the above regenerated kidneys with 4%paraformaldehyde. The magnification is 400 times. In (a) of FIG. 6 , theregenerated glomeruli were observed at a portion indicated by anarrowhead.

(b) of FIG. 6 is a fluorescence micrograph showing the observationresult of the fluorescence of GFP in the same field of view as (a) ofFIG. 6 . The magnification is 400 times. In (b) of FIG. 6 , fluorescenceof GFP was observed at a portion indicated by an arrowhead. This resultindicates that the regenerated glomeruli are derived from thetransplanted kidney precursor cells.

Based on the above results, it became clear that the kidney regenerationcan be performed in vivo.

Experimental Example 6

(Replacement of Both Metanephric Mesenchyme and Ureteric Bud of MouseEmbryo)

<<Tissue-Specific Removal of Ureteric Bud>>

In the present experimental example, the iDTR mouse having a genotype ofSix2-Cre-ER^(tg/wt)HoxB7-Cre^(tg/wt) was used. First, in the same manneras in Experimental Example 1, the metanephros was extracted from themouse embryo and subjected to organ-culture. Subsequently, diphtheriatoxin was added to the medium to tissue-specifically remove the uretericbud.

<<Transplantation of Kidney Precursor Cells>>

Subsequently, in the same manner as in Experimental Example 2, 1×10⁵kidney precursor cells derived from the wild-type mouse weretransplanted into the metanephros of the mouse, from which the uretericbud was removed. Thereafter, the organ culture of the metanephros wascontinued for 5 days.

<<Tissue-Specific Removal of Metanephric Mesenchyme>>

Subsequently, tamoxifen and diphtheria toxin were added to the culturemedium for organ culture. As a result, in the Six2-expressing cell,which is a metanephric mesenchyme-specific marker, Cre-ER istranslocated into the nucleus and recombines to express the diphtheriatoxin receptor. As a result, cells of the metanephric mesenchyme werekilled by diphtheria toxin. As a result, the metanephric mesenchyme wastissue-specifically removed.

<<Transplantation of Kidney Precursor Cells>>

Subsequently, in the same manner as in Experimental Example 2, 1×10⁵kidney precursor cells derived from the wild-type mouse weretransplanted again into the metanephros of the mouse, from which themetanephric mesenchyme was removed. Thereafter, the organ culture of themetanephros was continued for 3 to 5 days.

<<Immunostaining>>

Subsequently, the metanephros after organ culture was fixed with 4%paraformaldehyde, and therefore a tissue section sample was prepared.Subsequently, the metanephros was immunostained with an anti-calbindinantibody for staining the ureteric bud and observed with a fluorescencemicroscope.

(a) of FIG. 7 is a representative photograph of the metanephros of apositive control. For the positive control, the diphtheria toxin was notadded in the stage of removing the ureteric bud, and the kidneyprecursor cells were not transplanted. Therefore, in the positivecontrol, tissues derived from the original ureteric bud were maintained.

(b) of FIG. 7 is a representative photograph of the metanephros of anegative control. For the negative control, the diphtheria toxin wasadded in the stage of removing the ureteric bud, and the kidneyprecursor cells were not transplanted. Accordingly, because in thenegative control, the ureteric bud was tissue-specifically removed, andthe kidney precursor cells were not transplanted, the ureteric bud waslost.

(c) of FIG. 7 is a representative photograph of the metanephros of atest group. In the test group, the ureteric bud was tissue-specificallyremoved, and the kidney precursor cells were transplanted. As a result,the ureteric bud was replaced by the transplanted kidney precursorcells, and the growth of tissues derived from the ureteric bud wasobserved.

Table 1 shows the results of measuring the number of ureteric bud tipsin the metanephros of the positive control (n=5), the negative control(n=5), and the test group (n=5).

TABLE 1 Positive Negative Test control control group Number of uretericbud tips 145.2 ± 22.2 73.2 ± 29.2 120.8 ± 23.8 (average value ± standarddeviation)

As a result, in the negative control, a decrease in the number ofureteric bud tips was observed compared to the positive control. Incontrast, in the test group, the same number of ureteric bud tips as thepositive control was measured. This result shows that both metanephricmesenchyme and ureteric bud can be replaced with the transplanted kidneyprecursor cells by the method of the present experimental example.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to provide atechnique for producing a kidney from kidney precursor cells.

The invention claimed is:
 1. A kidney production method comprising: (i)tissue-specifically removing kidney precursor cells in a metanephricmesenchyme of a metanephros of a first non-human animal; (ii)transplanting, into the metanephros, a kidney precursor cell derivedfrom a second non-human animal which is allogeneic or xenogeneic to thefirst non-human animal; (iii) advancing development of the metanephros,wherein the transplanted kidney precursor cell is differentiated andmetanephric mesenchyme is regenerated, (iv) tissue-specifically removingkidney precursor cells in a ureteric bud of the metanephros; (v)transplanting, into the metanephros, a kidney precursor cell derivedfrom the second non-human animal which is allogeneic or xenogeneic tothe first non-human animal; (vi) advancing development of themetanephros, wherein the transplanted kidney precursor cell isdifferentiated and ureteric bud is regenerated; and (vii) forming akidney by advancing development of the metanephros, wherein (i) to (vii)are performed in an order of (i), (ii), (iii), (iv), (v), (vi), (vii) orin an order of (iv), (v), (vi), (i), (ii), (iii), (vii), wherein thekidney precursor cell is selected from the group consisting of a kidneyprecursor cell isolated from the second non-human animal, a kidneyprecursor cell differentiated from mesenchymal stem cells isolated fromthe second non-human animal, a kidney precursor cell differentiated fromiPS cells produced from cells isolated from the second non-human animal,and a kidney precursor cell differentiated from ES cells produced fromcells isolated from the second non-human animal.
 2. The kidneyproduction method according to claim 1, wherein the kidney precursorcell derived from the second non-human animal is a cat cell.
 3. Thekidney production method according to claim 1, wherein the firstnon-human animal is a pig.
 4. The kidney production method according toclaim 1, wherein the first non-human animal is a mouse.
 5. The kidneyproduction method according to claim 1, wherein the first non-humananimal is a cat.
 6. The kidney production method according to claim 1,wherein a gene which assists kidney formation is introduced into thekidney precursor cells transplanted in step (ii) or step (v).